Therapies for treating hepatitis c virus infection
A liver, VX-222 technology, applied in antiviral agents, medical preparations containing active ingredients, pharmaceutical formulas, etc., can solve problems such as uncertain prospects
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Embodiment 1
[0224] Example 1: HCV Replicon Cell Assay Protocol
[0225] Cells containing the hepatitis C virus (HCV) replicon were maintained in DMEM containing 10% fetal bovine serum (FBS), 0.25 mg / ml G418 and appropriate supplements (medium A).
[0226] On day 1, replicon cell monolayers were treated with a trypsin:EDTA mixture, removed, and Medium A was diluted to a final concentration of 100,000 cells / ml. Place 10,000 cells / 100 μl in each well of a 96-well tissue culture plate and incubate overnight at 37° C. in a tissue culture incubator.
[0227] On day 2, compounds (in 100% DMSO) were serially diluted into DMEM containing 2% FBS, 0.5% DMSO and appropriate supplements (medium B). The final concentration of DMSO was maintained at 0.5% throughout the serial dilutions.
[0228] The medium on the replicon cell monolayer was removed, and then medium B containing various concentrations of the compound was added. Medium B without any compound was added to other wells as no compound cont...
Embodiment 2
[0233] Embodiment 2: HCV Ki assay scheme
[0234] HPLC microbore method for separation of 5AB substrate and product
[0235] Substrate: NH 2 -Glu-Asp-Val-Val-(α)Abu-Cys-Ser-Met-Ser-Tyr-COOH SEQ ID NO:1.
[0236] Prepare a 5AB stock solution at a concentration of 20 mM (or concentration of choice) in DMSO w / 0.2M DTT. It was stored in aliquots at -20°C.
[0237] Buffer: 50 mM HEPES (pH 7.8); 20% glycerol; 100 mM NaCl.
[0238] The total assay volume was 100 μL.
[0239]
X1 (μL)
concentration in the assay
buffer
86.5
as above
5mM KK4A
0.5
25μM
1M DTT
0.5
5mM
DMSO or inhibitors
2.5
2.5%v / v
50 μM tNS3
0.05
25nM
[0240]
X1 (μL)
concentration in the assay
250 μM 5AB (priming)
20
25μM
[0241] Combine buffer, KK4A, DTT, and tNS3; add 78 μL each to each well of a 96-well plate. This was incubated at 30°C for about 5-10 minutes. 2.5 μL ...
Embodiment 3
[0257] VX-950 was tested in a randomized double-blind placebo-controlled single dose escalation study. 25 healthy male volunteers were recruited. Subjects each received multiple single doses of VX-950 (at least 7 days apart), 3 doses of VX-950 (increased dose levels), and 1 dose of placebo.
[0258] Doses from 25 mg to 1250 mg were evaluated. A dose escalation protocol was used that combined dose doubling and Fibonacci adjusted to be aggressive in the lower dose range and conservative in the higher dose range.
[0259] VX-950 was well tolerated at all dose levels and no serious adverse events were reported during the study. There was no increase in adverse events as dose levels were increased.
[0260] Pharmacokinetic analysis was performed using the statistical moment method. Pharmacokinetic analysis indicated that VX-950 was 最大 Be absorbed for 3 hours. Less than 2% of VX-950 was eliminated unchanged in urine, suggesting that the drug is primarily eliminated through met...
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